Metformin augments the levels of molecules that regulate the expression of the insulin-dependent glucose transporter GLUT4 in the endometria of hyperinsulinemic PCOS patients.

STUDY QUESTION: Does treatment with the insulin sensitizer metformin modify the levels and activation of proteins related to the expression of the insulin-dependent glucose transporter (GLUT4), such as adenosine monophosphate-activated protein kinase (AMPK) and myocyte enhancer factor 2A (MEF2A), in endometria from hyperinsulinemic hyperandrogenemic polycystic ovary syndrome (PCOS h-Ins) patients? SUMMARY ANSWER: In PCOS h-Ins patients, metformin increases endometrial levels of GLUT4 mRNA and protein levels by normalizing the quantity and activation of molecules that regulate GLUT4 expression to healthy values. These changes could improve endometrial metabolic function. WHAT IS ALREADY KNOWN: PCOS is an endocrine-metabolic disorders closely associated with insulin resistance. In particular, the insulin signaling pathway is impaired in endometria from these patients and the concentration of GLUT4, as well as the molecules involved in its translocation to the cell surface, is decreased. However, there are limited data about the mechanisms that regulate the GLUT4 expression in the endometria and the effect of metformin on them. STUDY DESIGN, SIZE AND DURATION: This is a case-control study in the setting of a research unit, approved by the Ethical Committees of our institution. The groups whose endometria were studied were PCOS h-Ins (n = 8); PCOS patients with hyperandrogenemia hyperinsulinemia taking only metformin for at least 3 months (PCOS-MTF, n = 8) and healthy fertile women at the time of hysterectomy because of benign pathology as controls (CE, n = 8). PARTICIPANTS/MATERIALS, SETTING, METHODS: Steroids and sex hormone-binding globulin were measured and glucose and insulin levels were evaluated during an oral glucose tolerance test. Protein levels for αAMPK (catalytic subunit of AMPK), phosphorylated (p)-AMPKαThr(172) (activating phosphorylation site), MEF2A, p-MEF2AThr312 (activating phosphorylation site) and GLUT4 were assessed by western blot and immunohistochemistry. In addition, GLUT4 gene expression was evaluated by RT-PCR. MAIN RESULTS AND THE ROLE OF CHANCE: We found significantly lower levels of MEF2A and p-MEF2AThr312 in PCOS h-Ins compared with CE endometria (P < 0.05). Also, we detected lower levels of p-AMPKαThr(172) in PCOS h-Ins endometria compared with the PCOS-MTF group (P < 0.05). The ratios of phospho-AMPK/total AMPK and phospho-MEF2A/total MEF2A were significantly increased in the PCOS-MTF compared with the PCOS h-Ins group (P < 0.05). The RT-PCR experiments showed lower levels of GLUT4 mRNA transcripts in PCOS h-Ins compared with PCOS-MTF-treated group (P < 0.05), the protein levels of GLUT4 were decreased in a similar way. LIMITATIONS, REASONS FOR CAUTION: The limited number of patients included in this study who presented large clinical variability. Therefore, it would be necessary to recruit a greater number of patients to minimize our data dispersion in order to prove the clinical benefits of metformin described by others. WIDER IMPLICATIONS OF THE FINDINGS: Since the insulin sensitizer metformin increases the expression of the GLUT4, it may improve endometrial physiology in PCOS patients and, therefore, promote better reproductive outcomes. These results suggest that in PCOS patients, metformin may act directly at the endometrial level and decrease insulin resistance condition by increasing the expression of GLUT4 and, in this way, indirectly restore endometrial function. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Fondo Nacional de Desarrollo Científico y Tecnológico (grant number 1095127 to M.V.). None of the authors has any conflict of interest to declare.

Changes in the expression of insulin signaling pathway molecules in endometria from polycystic ovary syndrome women with or without hyperinsulinemia.

Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder associated with insulin resistance and compensatory hyperinsulinemia. Scarce information is available on the expression of molecules involved in the insulin pathway in endometria from women with PCOS. Therefore, we examined the protein levels of insulin-signaling molecules, like insulin receptor, insulin-receptor substrate (IRS)-1, pIRS-1Y612, Akt, AS160, pAS160T642 and GLUT4 in endometria from PCOS women with or without hyperinsulinemia. Protein levels were assessed by Western blot and immunohistochemistry in 21 proliferative-phase endometria from control women (CE = 7), normoinssulinemic PCOS women (PCOSE-NI = 7) and hyperinsulinemic PCOS women (PCOSE-HI = 7). The data show no differences in the expression of insulin receptor between all groups as assessed by Western blot; however, IRS-1 and pIRS-1Y612 were lower in PCOSE-HI than controls and PCOSE-NI (P < 0.05). AS160 was detected in all analyzed tissues with similar expression levels between groups. Importantly, PCOSE-HI exhibited lower levels of pAS160T642 (P < 0.05) and of GLUT4 (P < 0.05) compared with CE. The immunohistochemistry for insulin receptor, IRS-1, Akt, AS160 and GLUT4 showed epithelial and stromal localization; IRS-1 staining was lower in PCOSE-HI (P < 0.05). In conclusion, human endometrium has the machinery for glucose uptake mediated by insulin. The diminished expression of GLUT4, as well as the lower level of pIRS-1Y612 and pAS160T642 exhibited by PCOSE-HI, suggests a disruption in the translocation of vesicles with GLUT4 to the cell surface in these patients.

Role of the transcriptional factors FOXO1 and PPARG on gene expression of SLC2A4 in endometrial tissue from women with polycystic ovary syndrome.

Fifty to seventy percent of patients with polycystic ovary syndrome (PCOS) present hyperinsulinemia. On the other hand, reports indicate that forkhead box class O 1 (FOXO1) and peroxisome proliferator-activated receptor-gamma (PPARG) are involved in the insulin signaling pathway, regulating the gene expression of SLC2A4 (GLUT4). The negative effect of FOXO1 over PPARG transcription disappears when FOXO1 is phosphorylated (p-FOXO1) and excluded from the nucleus, whereas PPARG can suppress gene expression of SLC2A4. Scarce knowledge is available in endometrium of women with PCOS and hyperinsulinemia (PCOSE h-Ins) about the role of these factors. We aimed to evaluate whether the endocrine and metabolic status of PCOS modify the levels of gene and protein expression of FOXO1, PPARG, and SLC2A4 in the endometria from hyperinsulinemic PCOS women compared with controls. In endometria from control (CE, n=7) or PCOSE h-Ins (n=7), we determined the subcellular location and protein levels of p-FOXO1Ser319 and FOXO1/FOXO4 by immunohistochemistry and western blot respectively; gene and/or protein levels of PPARG and SLC2A4 were evaluated by RT-PCR and/or western blot. Cytoplasm location for FOXO1 and p-FOXO1Ser319 was immunodetected in both groups of endometria, showing significantly higher staining in PCOSE h-Ins for these proteins (P<0.05). In PCOSE h-Ins, gene and protein levels of PPARG were significantly higher than in CE, whereas SLC2A4 mRNA was decreased (P<0.05). In conclusion, the derepression of PPARG transcription by the high levels of p-FOXO1Ser319 could partially account for the lower levels of SLC2A4 found in PCOSE h-Ins, suggesting an alteration of the endometrial function in these patients.

Preimplantation embryotoxicity after mouse embryo exposition to reactive oxygen species.

Exposure of either gametes or embryos to conditions and/or factors that generate oxidative stress has been associated with impaired early embryogenesis. The effects of reactive oxygen species (ROS) on mouse preimplantation development, depending of the ROS-concentration and time of exposition, were studied. Two-cell embryos were incubated with 5, 10, 25 and 50 microM of hydrogen peroxide (H2O2) for 30 and 60 minutes of exposition and allowed to develop for 72 h to study the quality of development. The incubation with 50 microM H2O2 for 30 or 60 minutes, strongly inhibited the 2-cell embryo development as compared to the control (p < 0.001). Twenty-five microM H2O2 produced inhibition of blastocyst formation (p < 0.001) and 10 microM H2O2 significantly decreased the percentages of expanded and hatched blastocysts, which resulted morphologically altered (p < 0.05 and p < 0.01, respectively). The higher H2O2 concentrations were able to elicit necrotic morphology in the 2-cell arrested embryos, while 10 microM H2O2 induced moderate damage with the arrested embryos partially fragmented. In conclusion, important causes for defective preimplantation development and for early embryo losses may be due to oxidative stress because early mouse embryos exposed to ROS for short times arrested at the first cellular cycle (2-cell) and/or impaired embryo differentiation and morphogenesis, being these effects ROS-concentration-dependent.

Hormonal profile and endometrial morphology in letrozole-controlled ovarian hyperstimulation in ovulatory infertile patients.

OBJECTIVE: To evaluate the clinical response and endometrial morphology during the implantation window on ovarian hyperstimulation with the aromatase inhibitor letrozole in infertile ovulatory women. DESIGN: Prospective trial in infertile patients. SETTING: Tertiary care hospital. PATIENT(S): Eight ovulatory infertile patient candidates for ovarian superovulation. INTERVENTION(S): Subjects were monitored in one control cycle. In the next cycle, they received letrozole 5.0 mg daily on days 3 through 7 after menses. MAIN OUTCOME MEASURE(S): Number of ovulatory follicles; dominant follicle diameter; endometrial thickness; hormonal profile of FSH, LH, E(2), A, T, and P; endometrial histological dating; and pinopode formation assessed by scanning electron microscopy. RESULT(S): Cycles stimulated with letrozole resulted in more ovulatory follicles than did natural cycles (mean +/- SD 2.0 +/- 0.9 vs. 1.0 +/- 0.0), which attained a greater preovulatory diameter (mean +/- SD 23.8 +/- 2.7 vs. 19.3 +/- 2.1 mm), with similar endometrial thickness at midcycle compared with spontaneous cycles. Endocrine profile of medicated cycles was characterized on day 7 by increased levels of LH (5.9 +/- 0.8 vs. 3.5 +/- 0.4 IU/mL), reduced E(2) (98.4 +/- 11.4 vs. 161.5 +/- 14.7 pmol/L), and elevated androgens. Preovulatory and midsecretory E(2) were similar to spontaneous cycle, and P levels during midluteal phase were significantly elevated (44.2 +/- 4.6 vs. 27.7 +/- 4.6 pmol/L). Endometrial morphology during the implantation window in letrozole-stimulated cycles was characterized by in-phase histological dating and pinopode expression on scanning electron microscopy. CONCLUSION(S): Letrozole induces moderate ovarian hyperstimulation in ovulatory infertile patients with E(2) levels similar to spontaneous cycles and higher midluteal P, leading to both a normal endometrial histology and development of pinopodes, considered to be relevant markers of endometrial receptivity.

Differential in vitro actions of nitric oxide on human endometrial cell survival.

OBJECTIVE: To investigate the presence of caspase-3 and Bcl-2 concentration in human endometrial tissue throughout the menstrual cycle, and study the effect of nitric oxide (NO) on cell proliferation and apoptosis during culture. DESIGN: Expression of caspase-3 and Bcl-2 concentration in endometrial explants, and examination of L-arginine (L-Arg) effect on epithelial and stromal cell proliferation and apoptosis in vitro. SETTING: Prospective study.Twenty-seven eumenorrheic women (37 +/- 1.2 years). INTERVENTION(S): Endometrial samples were obtained with Pipelle suction curette from the corpus of the uterus. MAIN OUTCOME MEASURE(S): Apoptosis (annexin V-FITC binding), Bcl-2 concentration (ELISA), caspase-3 (immunohistochemistry), cell proliferation (spectrophotometric assay), and gene expression (RT-PCR). RESULT(S): Caspase-3 was detected by immunoassay in epithelial tissue throughout the menstrual cycle and in stroma during secretory phase. The Bcl-2 concentration was similar in endometrial homogenates obtained throughout the menstrual cycle, but L-Arg decreased Bcl-2 only in endometrium from the proliferative phase. In epithelial cells, NO increased apoptosis by 2.1 +/- 0.2-fold, augmented mRNA expression of Bax, and reduced expression of Bcl-2 compared with basal cultures. In stromal cells, NO increased cell proliferation in a dose-dependent manner, an effect that was blocked by a NO synthase inhibitor. CONCLUSION(S): These data indicate that NO has a differential regulatory function on endometrial cell survival, as indicated by the results on stromal cell proliferation and epithelial cell apoptosis during culture, which suggests paracrine interactions between both cell types.

Absence of Y chromosome microdeletions in patients with cryptorchidism and hypospadias.

Microdeletions of the Y chromosome have been observed in some patients with cryptorchidism and severe defects of spermatogenesis. We investigated whether microdeletions of the Y chromosome may be present in patients with cryptorchidism and hypospadias. Peripheral blood was obtained from 20 male patients 5.8 +/- 4.1 years (range: 0.4-14 years) with cryptorchidism and hypospadias for somatic DNA analysis of Y chromosome using multiplex polymerase chain reaction. These patients had no identifiable genetic syndrome, other genitourinary malformations or an abnormal karyotype. We evaluated the presence or absence of amplification using a set of 34 different sequence-tagged sites (STS) in each patient. All patients showed normal length amplifications for each of the regions evaluated, suggesting that microdeletions of the Y chromosome are not a frequent cause of hypospadias associated with cryptorchidism.

Nerve growth factor induces vascular endothelial growth factor expression in granulosa cells via a trkA receptor/mitogen-activated protein kinase-extracellularly regulated kinase 2-dependent pathway.

CONTEXT: Acquisition of ovulatory competence by antral follicles requires development of an adequate vascular supply. Although it is well established that ovarian angiogenesis is cyclically regulated by vascular endothelial growth factor (VEGF), the factors controlling VEGF production by ovarian follicles remain largely unknown. Nerve growth factor (NGF) may be one of these factors, because NGF promotes angiogenesis and synthesis of angiogenic factors in other tissues and is produced by human granulosa cells (hGCs). OBJECTIVE: The aim of the study was to determine whether NGF influences the production of VEGF by hGCs and to identify a potential signaling pathway underlying this effect. DESIGN: We conducted a prospective experimental study. PATIENTS: hGCs were obtained from 41 women participating in the in vitro fertilization program of our institution. METHODS: Changes in VEGF mRNA after exposure to NGF were evaluated in cultured hGCs by PCR and real-time PCR. The effect of NGF on VEGF secretion was determined by ELISA. The involvement of trkA, the high affinity NGF receptor, was examined by inhibiting the receptor's tyrosine kinase activity with K252a. The contribution of an ERK1/ERK2-mediated signaling pathway was identified by detecting NGF-dependent phosphorylation of these proteins and by blocking their activity with the inhibitor U0126. RESULTS: NGF promotes VEGF production in cultured hGCs. Blockade of trkA receptor tyrosine kinase activity blocks this effect. NGF induces MAPK-ERK2 phosphorylation, and blockade of this signaling pathway prevents the NGF-induced increase in VEGF production. CONCLUSIONS: NGF promotes ovarian angiogenesis by enhancing the synthesis and secretion of VEGF from hGCs via a trkA- and ERK2-dependent mechanism.

Azoospermia y fertilización asistida: rol de la histología testicular en el éxito de icsi / Azoospermia and assisted reproduction: role of testicular histology in the success of ICSI

Introducción: La infertilidad masculina afecta aproximadamente al 7por ciento de los hombres, presentándose hasta el 15 por ciento de ellos con azoospermia. El conocimiento del tipo de azoospermia (obstructiva o no obstructiva) y la localización de la falla (pre-testicular, testicular o post-testicular) es vital para conocer el pronóstico de fertilidad de la pareja y plantear un plan terapéutico adecuado. Actualmente, la extracción de espermatozoides desde epidídimo o testículo de pacientes azoospérmicos, y la posterior inyección intracitoplásmática de éstos (ICSI, por sus siglas en inglés) ha permitido obtener embriones viables para su posterior transferencia. Materiales y métodos: Estudio descriptivo retrospectivo de 42 parejas infértiles con diagnóstico de azoospermia; que se sometieron a biopsia testicular, ICSI y posterior transferencia de embriones, entre los años 2004 y 2012. Se lleva a cabo un análisis de la edad de los pacientes, resultados de la histopatología testicular y su asociación con los resultados de la fertilización asistida. Resultados: 42 pacientes azoospérmicos se sometieron a biopsia testicular y extracción de espermatozoides en el mismo acto quirúrgico. La edad promedio de los pacientes fue de 36 años para los hombres y 32 años para las mujeres. En el análisis histológico de los tejidos testiculares, el 31por ciento de los pacientes presentaban espermatogénesis conservada (EC), el 35.7 por ciento atrofia mixta (AM), el 14.3 por ciento hipoespermatogénesis (HE), el 14.3 por ciento detención de la maduración (DM) y un 4.8 por ciento presentaba otras histologías. Lograron embarazo clínico 14 de 42 parejas (33,3 por ciento), siendo la tasa de embarazo específica por patología de 38,5 por ciento para EC, 26.7 por ciento para AM, 50 por ciento para HE, 16,7 por ciento para DM y 50 por ciento para las otras histologías. 12 de las 42 parejas realizaron más de un ciclo de transferencias...

Introduction: Male infertility affects approximately 7percent of men, presenting up to 15 percent with azoospermia. Knowing the type of azoospermia (obstructive or non-obstructive) and the location of the problem (pre-testicular, testicular and post-testicular) is vital to recognize the fertility prognosis of the couple and present a proper treatment plan. Currently, the extraction of sperm from epididymis or testis of azoospermic patients, and subsequent intracytoplasmic sperm injection (ICSI) has yielded viable embryos for transfer. Materials and Methods: Retrospective study of 42 infertile couples diagnosed with azoospermia, who underwent testicular biopsy, ICSI and subsequent embryo transfer, between 2004 and 2012. We performed an analysis of the patients’ age, testicular histopathology results and their association with assisted fertilization outcome. Results: 42 azoospermic patients underwent testicular biopsy and sperm extraction in the same surgery. The average age of patients was 36 years for men and 32 years for women. Histologic analysis of testicular tissue showed that 31 percent of patients had normal spermatogenesis (NS), 35.7 percent mixed atrophy (MA), 14.3 percent hypospermatogenesis (HS), 14.3 percent maturation arrest (MTA) and 4.8 percent had other histologies. 14 of 42 couples achieved clinical pregnancy (33.3 percent), with a specific pregnancy rate of 38.5 percent for NS, 26.7 percent for MA, 50 percent for HS, 16.7 percent for MTA and 50 percent for other histologies. 12 of 42 couples underwent more than one transfer cycle. Conclusions: The use of ICSI is a suitable alternative for infertile couples with severe male factor. The results of this technique are favorable for most histologies. Patients with MA exhibit sertoli solo syndrome and / or tubular sclerosis foci associated to regions with normal spermatogenesis, enabling the sperm extraction for ICSI.

Rendimiento de la microcirugía en hidrosalpinx: análisis de tablas de vida / Yield of hidrosalpinx microsurgery: analysis of life tables

Se analizan los resultados de la cirugía en hidrosalpinx mediante análisis de tablas de vida de 105 pacientes del Policlínico de Infertilidad del Hospital Clínicp San Borja Arriarán, operadas por vía laparotómica entre 1980-1990, con seguimiento hasta los 48 meses. (Sólo se consideran las pacientes que presentan como único factor de esterilidad un daño tubario dilatado y sometidas a microcirugía por primera vez). Se analizan y comparan los rendimientos según grupo etáreo, tipo y lateralidad del daño tubario, duración de la esterilidad y tipo de cirugía. Se obtuvo una tasa acumulativa de embarazo de 62 por ciento a los 48 meses de seguimiento y la probabilidad de embarazo por ciclo (fecundabilidad) fue de 1,5 por ciento. Estos resultados son comparables a los publicados en otras series nacionales y extranjeras